JPH0515435B2 - - Google Patents

Abstract

A method for producing interferon which comprises cultivating a microorganism carrying an expression vector with a structural gene of interferon inserted therein in a chemically defined medium containing L-glutamic acid and an iron ion source and recovering interferon from the culture, gives high yield of interferon.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing interferon. More specifically, the present invention provides a method for developing human interferon, which is characterized by culturing a microorganism carrying an expression vector incorporating a human interferon gene in a specific synthetic medium, accumulating a significant amount of interferon, and collecting the interferon. Regarding the manufacturing method of Elon. Interferon (hereinafter sometimes abbreviated as "IF") is a protein produced by cells of higher animals when stimulated by viruses, nucleic acids, etc., and has antiviral and antitumor effects. have It is currently known that there are three types of human IF, α-type, β-type, and γ-type, with different properties.The α-type and β-type are viruses and nucleic acids, and the γ-type is mitogenic. It is induced by diene etc. Therefore, the method of manufacturing it has been to culture human cells or established cell lines, but the production volume is extremely small, and the supply of the amount necessary for large-scale clinical trials and use as a therapeutic drug is insufficient. It is possible. However, in recent years, with advances in genetic manipulation technology,
All of the α, β, and γ types can now be relatively easily obtained as biologically active proteins from cultures such as Escherichia coli that carry expression vectors incorporating IF-producing genes [Nature
284, 316 (1980); Nature 287 , 411 (1980); Proceedings of the National Academy of Sciences of the United States of America
Sciences of the United States of America,
Hereinafter, it will be abbreviated as Proc.Nat.Acad.Sci.USA. ) 77 , 5230 (1980); Nucleic Acids Research 8 ,
4057 (1980); Nature 295 , 503 (1982)]. However, the production volume is not necessarily sufficient for industrial human IF production. In view of this situation, the present inventors have repeatedly investigated various methods for culturing microorganisms that have expression vectors incorporating IF-producing genes. However, it was discovered that a significantly higher yield of IF could be obtained by converting this into a specific completely synthetic medium and culturing it.Based on this, further research led to the completion of the present invention. . The present invention involves culturing a microorganism carrying an expression vector incorporating an interferon-producing gene in a synthetic medium containing L-glutamic acid and an iron ion source with known components, and collecting interferon from the culture. This is a unique method for producing interferon. Three types of human IF are known: α type, β type, and γ type. In particular, there are many molecular types of α type, including A, B, C, D, F, H, I, and J. It is known that the gene has been cloned and expressed in E. coli (see Japanese Patent Application Publication No. 79897/1989 and European Patent Application Publication No. 43980). Also β
Type IF gene (see Japanese Patent Application Publication No. 140793/1983)
and γ-type IF genes (see Japanese Patent Application Publication No. 58-90514) have also been expressed in Escherichia coli.
Any of these genes or other human IFs
The present invention can be applied to the production of any IF, even if it is a gene, as long as it is expressed in a host microorganism. In order to efficiently express the IF gene in a host microorganism, especially E. coli, ColEI-derived pBR 322 [Gene 2 , 95 (1977)] is most often used as the expression vector plasmid, but other Any plasmid that can be replicated and maintained in E. coli can be used. Examples include pBR313 [Gene 2, 75 (1977)], pBR324, pBR325 [Gene 4 ,
121 (1978)], pBR327, pBR328 [Gene 9 , 287
(1980)], pKY2289 [Gene 3 , 1 (1987)],
pKY2700 [Biochemistry 52 , 770 (1980)], pACYC177,
pACYC184 [Journal of Bacteriology 134 , 1141
(1978)], pRK248, pRK646, pDF41 [Methods in Engineering
Enzymology) 68 , 268 (1979)]. In addition, bacteriophages such as λgt and λC of the λgt system using λphage [Proc.Nat.Acad.
Sci.USA71, 4579 (1974)], λgt・λB [Proc.
Nat.Acad.Sci.USA 72 , 3416 (1975)], λDam
[Gene 1 , 255 (1977)] and Sharon Vector [Science 196 , 161 (1977); Journal of Virology]
Virology) 29 , 555 (1979)], vectors using filamentous phage, etc. can also be used as expression vectors. The IF gene is preferably linked downstream of a promoter, and the promoter includes:
Tryptophan (trp) promoter, lactose (Iac) promoter, protein chain elongation factor Tu
(tufB) promoter, recA gene promoter, λP _L and λP _R promoters involved in the proliferation of λ phage, and any of these promoters may be used. An expression vector incorporating an IF-producing gene may be constructed according to a known method. For example, regarding α-type IF, Nature
Vol. 287, p. 411 (1980), DNA Vol. 1, p. 125 (1982), New York Institute Research Vol. 11, p. 2927 (1983), Japanese Patent Application Publication No. 1987-79897
Publication (European Patent Application Publication No. 43980)
Regarding the method described in, for example, β-type IF, see New York Assists Research Vol. 8, p. 4057 (1980), Proc. Nat. Acad. Sci. USA, Vol. 77, 5230.
Page (1980). New York Assists Research Vol. 11, p. 4677 (1983), Japanese Patent Application Publication No. 1987-
Regarding the method described in Publication No. 140793, etc., and γ-type IF, see Nature Vol. 295, p. 503 (1982), Japanese Patent Application Publication No. 1982-90514, and Japanese Patent Application Publication No. 1989-189197.
No. Publication, Japanese Patent Application No. 1982-176090 (Japanese Unexamined Patent Publication No. 1983-186995), Japanese Patent Application No. 1983
Specification No. -45723 (Japanese Unexamined Patent Publication No. 169494/1983)
Examples include the method described in . Escherichia coli is used as a host bacterium into which an expression vector incorporating an IF production gene is introduced, and among them, one derived from E. coli strain K-12 is particularly preferred from the viewpoint of handling and safety. Examples of those derived from the E. coli K-12 strain include E. coli 294 strain,
Examples include W31110 strain, C-600 strain, and X1776 strain. Moreover, these mutant strains may also be used. The above 294 strains include Proc.Nat.Acad.Sci.US
A. 73 , 4174 (1976). In addition, the 294 strains mentioned above include The American Type Culture Collection (The
American Type Culture Collection, hereinafter
It is abbreviated as ATCC. ) Catalog of Strains 15th edition 1982
One example is the strain listed as ATCC 31446. Furthermore, examples of the above-mentioned 294 strains include the strain deposited with the Fermentation Research Institute as IFO 14171 on March 23, 1982 (see Japanese Patent Application Publication No. 189197/1983). The above W31110 strain is ATCC 27325 in ATCC Catalog of Strains 15th edition 1982.
Stocks listed as . The above C-600 strain is ATCC 23724 in ATCC Catalog of Strains 15th edition 1982.
Stocks listed as . The above X1776 strain is The Journal of Infectious Daisy
Journal of Infectious Diseases) 137 , 668
(1978). Examples of the X1776 strain include the strain listed in US Pat. No. 4,190,495 as ATCC 31244 (published in ATCC Catalog of Strains, 15th edition, 1982). An expression vector (plasmid, vector, or phage vector) incorporating an IF-producing gene may be introduced into a host bacterium by any known method. The method includes, for example, journal
Journal of Molecular Biology
Examples include methods described in Molecular Biology) 53 , 159 (1970), Merits in Engineering 68 , 253 (1979), Gene 3 , 279 (1978), and the like. The synthetic medium used in the present invention refers to one whose components are all known. The synthetic medium may be, for example, a known inorganic salt-based medium [eg, M-9 medium (see Table 1 below),
Davis medium, etc.], but TSM-3 medium having an inorganic salt composition shown in Table 3 below can also be advantageously used. As the seed culture medium, ordinary natural media such as nutrient broth or L-broth may be used, or synthetic media (SS
-1 medium) can also be used more advantageously. [Table] [Table] [Table] [Table] L-glutamic acid, an iron ion source, a zinc ion source and a copper ion source used in the method of the present invention are each added in a state in which the components are known. L-glutamic acid used in the method of the present invention may be in the form of a salt. Examples of such salts include sodium salts, potassium salts, ammonium salts, and the like. The amount of L-glutamic acid or its salt added is about 0.1 to 1 liter of L-glutamic acid per liter of synthetic medium.
It is 10g. The iron ion source used in the method of the present invention refers to a substance that becomes iron ions when made into a solution or a substance that is used in the form of iron ions. Examples of the iron ion source include ferrous chloride, ferric chloride,
Examples include ferrous sulfate, ferric sulfate, ferric phosphate, ferric nitrate, ferric citrate, and ferrous lactate. The amount of the iron ion source added is about 10 ^-5 to 10 ^-3 mol of iron ions per liter of synthetic medium. In the present invention, it is advantageous to further add a zinc ion source, a copper ion source, or a zinc ion source and a copper ion source to the synthetic medium because the yield of the target product may be further increased. The above-mentioned zinc ion source refers to a substance that becomes zinc ions when made into a solution or a substance that is used in the form of zinc ions. Examples of the zinc ion source include zinc chloride, basic zinc carbonate, zinc nitrate, zinc sulfate, zinc phosphate, and the like. The amount of the zinc ion source added is about 10 ^-5 to 10 ^-3 mol of zinc ions per liter of synthetic medium. The above-mentioned copper ion source refers to a substance that becomes copper ions when made into a solution or a substance that is used in the form of copper ions. Examples of the copper ion source include copper sulfate, cupric chloride, cuprous chloride, copper carbonate, copper acetate, and the like. The amount of copper ion source added is approximately 1 liter of copper ion per liter of synthetic medium.
10 ^-5 to 10 ^-3 mol. In addition, when the host bacteria exhibits auxotrophy, the required amino acids (e.g. L-lysine, L-arginine,
L-methionine, L-leucine, L-proline,
It is necessary to add L-isoleucine, L-valine, L-tryptophan, etc.) at a rate of about 10 to 1000 mg. Vitamins (e.g., calcium pantothenate, choline chloride, folic acid, i-
Inositol, nicotinamide, pyridoxal hydrochloride, riboflavin, vitamin B ₁ , etc.) are not particularly necessary unless a vitamin auxotrophic mutant strain is used, but fermentation tends to be stabilized by adding about 1 to 100 mg of vitamin B ₁ . It is desirable to add it as appropriate. In addition, if the host bacteria is auxotrophic for vitamins, the required vitamins will be about 1 to 100mg/
It is necessary to add The carbon source added to the medium was approximately 0.1 during the culture period.
It is advantageous to maintain the ratio between 5% and 5% (W/V) because a significant amount of the desired IF can be accumulated. Examples of the carbon source include glucose, glycerol, maltose, and sorbitol. Plasmids containing interferon genes are usually marked with antibiotic resistance selection marks, and in this case, addition of antibiotic resistance (e.g., tetracycline, ampicillin, etc.) to the culture medium will cause retention of the plasmid. It is advantageous to selectively propagate only those strains that have been tested. Culture can usually be performed by stirring culture. The oxygen concentration in the medium is approximately 5% of the saturated dissolved oxygen concentration.
It is advantageous to carry out the culture while maintaining the ratio above (V/V) because the production amount of the desired IF can be increased. For this purpose, it is also effective to mix pure oxygen with air and aerate it during the culture. In the culture of the present invention, the pH of the medium is usually preferably adjusted to about 5 to 7.5. The culture temperature is approx.
15 to 45°C, more preferably about 20 to 42°C
It is. Culture time is about 3 to 72 hours. During the fermentation of the present invention, IF is normally accumulated within the bacterial cells, so in order to collect the IF accumulated in the culture, the bacterial cells are first collected by centrifugation or filtration.
This is done by extracting the IF.
In order to efficiently extract IF, for example, ultrasonication, lysozyme treatment, treatment with chemicals such as surfactants, etc. are performed. The IF extracted in this way can be purified using conventional protein or peptide purification methods, such as ammonium sulfate salting out, alcohol precipitation, ion exchange column chromatography, cellulose column chromatography, and gel filtration. It is done. In particular, by combining monoclonal antibody methods, it is possible to obtain extremely pure specimens. That is, for example, a liquid subjected to an extraction operation is centrifuged to obtain a supernatant, which is applied to, for example, a monoclonal antibody column, and the column is washed with, for example, 0.2M acetic acid, 0.1% Triton x 100, and 0.15M NaCl. It elutes with In this operation,
Since interferon is specifically adsorbed to a monoclonal antibody column, a highly pure sample can be easily obtained. [For purification of α-type IF,
Scientific American
See American) 243 , 66 (1980). Regarding the purification of γ-type IF, see Japanese Patent Application No. 176091/1988 (see Japanese Patent Application Publication No. 80646/1988). For example, the human leukocyte IFαA protein obtained in this way can be used to treat vesicular stomatitis against MDBK cells derived from Ui kidney. In antiviral activity measurement by virus (VSV) cytopathic effect inhibition test, 10 ⁸ U/
Shows specific activity of mg or more. In addition, human immune IF protein has an antiviral activity of 10 ⁷ U/mg in a test to inhibit the cytopathic effect of vesicular stomatitis virus (VSV) on human amnion-derived WISH cells.
[Japanese patent application 176091/1989]
(Refer to Japanese Patent Application Laid-open No. 59-80646.) The human IF proteins produced by the present invention are
The physicochemical and biological properties are exactly the same as those obtained by culturing the same recombinant in a natural medium. Therefore, produced by the method of the present invention
The IF can be used for the same purpose and in the same manner as IF produced by conventional methods. IF has antiviral effects, antitumor effects, cell growth inhibition effects, immunosuppressive effects, etc.
IF is a mammal (e.g., human, bovine, equine, porcine,
It can be used to treat viral infections and tumors in mice, rats, etc.). For example, humans
IF antiviral agents, antitumor agents, cell growth inhibitors,
To use it as an immunosuppressant, for example, IF is mixed with known pharmacologically acceptable carriers, excipients, diluents, etc., and administered as an injection parenterally by intravenous or intramuscular injection. . The dosage is about 100,000 to 100 million units per day for a normal person, preferably about 1 million to 50 million units. In addition, the dosage for mammals other than humans is 2000
The amount is about 20,000 to 2 million units/Kg/day, more preferably about 20,000 to 1 million units/Kg/day. The present invention will be explained in more detail below by giving experimental examples and examples. Experimental Example 1 In the M-9 medium or TSM-3 medium described above,
Glucose was added as a carbon source at a rate of 10 g/E. coli 294 in a medium with and without monosodium glutamate added.
(ATCC31446)/pLe IF A trp 25 strains (JP-A-Sho
Publication No. 57-79897, European Patent Application Publication No.
43980) was inoculated and cultured at 37°C for 16 hours, and its growth was examined, and the results shown in Table 5 were obtained. [Table] Experimental Example 2 A medium 2.5 in which 25 g/glucose and 4 g/sodium L-glutamate were added to TSM-3 medium was placed in a 5-volume jar fermenter, and each of the metal salts listed in Table 6 was added thereto. E. coli 294 (ATCC31446)/pLe containing a plasmid containing the human leukocyte IF-αA gene was added to the culture medium.
50 ml of a seed culture of IF A trp 25 strain was inoculated and cultured at 37° C. with aeration rate of 2.5/min, stirring at 1000 rpm.
During culturing, as the bacteria grow, the culture temperature is successively lowered from 37°C to 33°C, 29°C, and 25°C, and furthermore, when the glucose concentration drops to 1% or less during culturing.
Freshly added 2.5% glucose and continued culturing for 27 hours. In addition, the pH during culture is adjusted using ammonia water.
I kept it at 6.8. The growth of the bacteria and the production amount of human leukocyte interferon αA at that time were examined, and the results shown in Table 6 were obtained. [Table] Experimental Example 3 L-seed medium in Table 7 or SS-1 described above
Pour 50ml of seed medium into a 200ml Erlenmeyer flask,
After adding 5 mg/tetracycline hydrochloride to this, E. coli 294 (ATCC31446)/pLe IF containing a plasmid incorporating the human leukocyte IF-αA gene was added.
A trp 25 was inoculated and cultured at 37°C for 12 and 16 hours, respectively. [Table] Next, 25 g/glucose, 4 g/sodium L-glutamate, 27 mg/feCl ₃ 6H ₂ O, 8 mg/glucose were added to the TSM-3 medium described above.
CuSO ₄・5H ₂ O, 8 mg/ZnSO ₄・7H ₂ O, 70
2.5 mg/ml of thiamine hydrochloride and 5 mg/ml of tetracycline hydrochloride were added to a 5-volume jar fermenter, and the above seed culture was inoculated into this and cultured under the same conditions as Experimental Example 2. The productivity of leukocyte IF was measured and the results shown in Table 8 were obtained. [Table] As is clear from Table 8, it is better to use a seed culture of a synthetic medium (SS-1 seed medium) than to use a seed culture of a natural medium (L-seed medium) in the main fermentation. It can be seen that the proliferation of IF is accelerated and the amount of IF produced is increased. Experimental Example 4 50ml of SS-1 seed medium was placed in a 200ml Erlenmeyer flask, and after adding 5mg/tetracycline hydrochloride to it, Escherichia coli 294 containing a plasmid incorporating the human leukocyte IF-αA gene was prepared.
(ATCC31446)/pLe IF A trp 25 strain was inoculated and cultured at 37°C for 16 hours. Next, add 4 g/sodium L-glutamate to TSM-3 medium, 27
mg/ferric chloride, 8 mg/copper sulfate, 8 mg/
of zinc sulfate, 70 mg of thiamine hydrochloride and 5
Medium supplemented with mg/mg of tetracycline hydrochloride
Put 2.5 pieces into a 5 volume jar fermenter,
Glucose was added to this under the conditions shown in Table 9.
After inoculating the above seed culture, the aeration rate was 2.5/min.
Culture was started at 37℃ with stirring at 1000 rpm, and as the bacteria grew, the culture temperature was gradually lowered from 37℃ to 33℃, 29℃, and 25℃ until the dissolved oxygen concentration was 10% of the saturated oxygen concentration. I kept it as high as possible. PH during culture
The pH was adjusted to 6.8 with aqueous ammonia, and culture was continued for 27 hours. Bacterial growth and human white blood cells at that time
The production amount of IF-αA was investigated and the results shown in Table 9 were obtained. [Table] [Table] As is clear from Table 9, Experiment No. 4-1, 4-
In the case of 2,4-3, bacterial growth and IF-αA
It can be seen that the productivity is extremely high. Example 1 Escherichia coli 294 (ATCC31446)/pLe IF carrying an expression plasmid incorporating the human leukocyte IF-αA gene
A trp 25 strain was added to (1) M-9 medium with glucose 5
g/, casamino acid 5 g/, vitamin B ₁ hydrochloride 70 mg/ and tetracycline hydrochloride 5 mg/
Added medium (natural medium) or (2) M-9 medium containing glucose 25g/, sodium L-glutamate 4g/, FeCl ₃ 6H ₂ O 27mg/,
CuSO ₄・5H ₂ O, 8 mg/, ZnSO ₄・7H ₂ O 8
mg/, vitamin ₁ hydrochloride 70 mg/, tetracycline hydrochloride 5 mg/, L-proline 50 mg/, and L-leucine 50 mg/. 2.5/min, stirring 1000r.p.
m., Started culturing at 37℃, halfway at OD3000KU at 33℃.
The temperature was lowered to 29°C for 5000KU and 25°C for 7000KU, and culture was continued for 48 hours. The enzyme concentration in the culture was maintained at 5% or higher. When the glucose concentration in the culture solution decreased to 1% or less during the course of the culture, glucose was added at a rate of 25 g/g. The results are shown in Table 10. [Table] Expressed as relative values when
As is clear from Table 10 above, (2) IF productivity was significantly increased when a synthetic medium containing the compound of the present invention was used. Example 2 Synthetic medium culture solution 2 (2) obtained in Example 1 was centrifuged to collect bacterial cells, which were mixed with 100ml of 10% sucrose, 0.2M NaCl, 10mM ethylenediaminetetraacetate (EDTA), 10mM 50mM with spermidine, 2mM phenylmethylsulfonyl fluoride (PMSF), 0.2mg/ml lysozyme
The suspension was suspended in Tris-HCl (PH7.6), stirred at 4°C for 1 hour, kept at 37°C for 5 minutes, and then treated with an ultrasonic crusher (Artek, USA) at 0°C and 40°C.
Processed in seconds. This lysate was centrifuged at 11,300× for 1 hour, and 95 ml of supernatant was collected. 95ml of this supernatant was mixed with 300ml of 20mM Tris-HCl (PH7.6) (TEN) containing 1mM EDTA and 0.15M NaCl.
After diluting to
I put it on. After thorough washing with TEN, IF-αA was further eluted with 0.2M acetic acid containing 0.1% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.), the active fraction was collected, adjusted to pH 4.5, and applied to a CM cellulose column. After adsorption and sufficient washing, elution was performed with 0.025M ammonium acetate buffer (PH5.0) containing 0.15M NaCl. The active fraction was collected again and lyophilized to yield 320 mg of human leukocytes.
IF-αA powder was obtained. The molecular weight of this product determined by SDS-polyacrylamide gel electrophoresis was 19,000±1,000.
Further, the specific activity of the final human leukocyte IF protein obtained here was 2×10 ⁸ U/mg. Also,
In terms of other physicochemical properties, amino acid composition, and peptide mapping, it exhibited exactly the same behavior as recombinant human leukocyte IF produced in a conventional medium. Example 3 Escherichia coli 294 (IFO 14171)/pHIT・trp carrying an expression plasmid incorporating a human immunity IF gene
(1) TSM
-3 medium glucose 25g/yeast extract 20
(2) TSM-3 medium with glucose 25g/ and sodium L-glutamate 4
g/, FeCl ₃・6H ₂ O 27 mg/, CuSO ₄・
5H ₂ O 8mg/, ZnSO ₄・7H ₂ O 8mg/,
Medium supplemented with thiamine hydrochloride 70 mg/ and tetracycline hydrochloride 5 mg/2.5 each (synthetic medium)
The culture was started at 37℃ with aeration of 2.5/min and agitation of 1000 rpm, and when the OD reached 2000 cret units, it reached 33℃ and reached 4000 cret units. When the temperature reached 29℃,
When the volume reached 6,000 Cret units, the temperature was lowered to 25°C and cultured for 26 hours. The pH of the culture solution is 6.8 with ammonia water.
When the glucose concentration in the culture solution decreased to 1% or less, glucose was added at a rate of 25 g/g. As a result, if the IF-γ productivity in the natural medium (1) was 100, the productivity in the synthetic medium (2) was 550. Example 4 The synthetic medium culture solution 2.4 obtained in Example 3 was centrifuged to collect bacterial cells, which were mixed with 120ml of 10% sucrose, 10mM EDTA, 10mM spermidine,
50mM with 2mM PMSF, 0.2mg/ml lysozyme
Suspended in Tris-HCl (PH7.6), stirred at 4℃ for 1 hour, kept at 37℃ for 5 minutes, and further treated with an ultrasonic crusher (Artek, USA) at 0℃ for 40 seconds. did. This lysate was centrifuged at 11,300×g for 1 hour, and 115 ml of supernatant was collected. After diluting 115 ml of this supernatant to 360 ml with TEN,
Anti-IF-γ antibody column (25ml) [Japanese patent application filed in 1980]
See Examples 12 and 13 of specification 176091. ], washed thoroughly with TEN, and then washed with 1M NaCl,
20mM Tris−HCl (PH
7.0) and then Tris-HCl (PH
7.0) and 100ml of the obtained active fraction was eluted with 0.115
% _Na2HPO4 , 0.02% _KH2PO4 , _0.8 % _NaCl , 0.02
Dialysis was performed at 4° C. for 18 hours against a buffer consisting of % KCl. The final amount of human immune IF protein obtained here was 47 mg, and its specific activity was 2×10 ⁷ U/mg. In addition, the molecular weight of the standard sample obtained here was determined to be 18000± by SDS-polyacrylamide electrophoresis.
1000, and in other physical and chemical properties, amino acid composition, and peptide mapping, it exhibited exactly the same behavior as the recombinant human immune IF produced in a conventional culture medium.

Claims (1)

[Scope of Claims] 1. A microorganism carrying an expression vector incorporating an interferon-producing gene is cultured in a synthetic medium containing L-glutamic acid and an iron ion source with known components, and interferon is collected from the culture. A method for producing interferon characterized by: 2 Claim 1, characterized in that a zinc ion source is further added to the medium in a state where the ingredients are known.
Manufacturing method described in section. 3. The manufacturing method according to claim 1 or 2, characterized in that a copper ion source is further added to the medium in a state in which the ingredients are known. 4 Claim 1 in which the interferon-producing gene is a human leukocyte interferon gene
The manufacturing method described in Items 1 to 3. 5. The production method according to claims 1 to 3, wherein the interferon-producing gene is a human immune interferon gene. 6 L-glutamic acid about 0.1 to 10g/,
6. The production method according to claims 1 to 5, wherein about 10 ^-5 to 10 ^-3 mol/of iron ion source is added. 7. The manufacturing method according to claims 2 to 6, wherein about 10 ^-5 to 10 ^-3 mol/zinc ion source is added. 8. The manufacturing method according to claims 3 to 7, wherein about 10 ^-5 to 10 ^-3 mol/copper ion source is added. 9. The production method according to claims 1 to 8, wherein the culture is carried out while maintaining the carbon source in the medium at about 0.1 to 5%. 10. The production method according to claims 1 to 9, wherein the culture is carried out while maintaining the oxygen concentration in the medium at about 5% or more of the saturated dissolved oxygen concentration.